Lipid emulsions are widely used in the treatment of patients who are not able to obtain adequate nutrition from their oral intake. These emulsions consist of (1) an oil, comprised of triglycerides, the primary nutritional component of the emulsion, (2) phospholipid as the emulsifying agent, and (3) glycerol to render the emulsion isotonic to the blood into which the emulsion is infused. Substantial benefits in the nutrition of these patients have resulted from the availability of lipid emulsions, through their provision of essential fatty acids and calories contained mainly in the oil.
The need for an emulsifying agent in these clinically used lipid emulsions results from the fact that the oil (e.g., soybean, safflower) cannot be dissolved or suspended in the aqueous solution required for intravenous infusion. The use of lecithin, a phospholipid, to form a stable emulsion of the oil comes from the knowledge that when phospholipids are sonicated in aqueous solutions, they become micelles which remain in suspension and also can hold an oil in suspension. Other natural compounds, such as cholesterol, have also been used, in combination with phospholipids such as lecithin, in laboratory studies to form micelles, but cholesterol or other sterols have never been used as emulsifying agents in lipid emulsions for human i.v. infusion.
Separately from inhibitory effects on cellular functions of the lipid emulsion which are used in clinical practice (discussed in the next paragraph), micelles of lecithin and/or cholesterol have been the subject of laboratory studies directed toward understanding the exchange of cell membrane lipids with their extracellular environment. These studies have demonstrated that the lipid composition of the extracellular environment can alter the lipid composition of the cell membrane and modulate certain cellular processes including cell proliferation. Exposure of cells to lecithin micelles may interfere with certain cellular functions. Whether micelles composed of a combination of lecithin and cholesterol have less inhibitory effects has not yet been resolved in the published literature; some studies have shown less, and some studies equal, inhibition by micelles composed of lecithin and cholesterol versus lecithin alone. No studies of these lipids in combination with the oil used in lipid emulsions have been published.
The problem addressed by this invention is the fact that in recent years significant adverse effects of lipid emulsions have been reported. Several medical journals have recommended great restriction in the use of lipid emulsions such as Intralipid .RTM. because of these side effects, Editorial "Parenteral nutrition in the newborn--a time for caution" Lancet ii: 838-839, 1980, which include, among others, suppressive effects on immune responses. These immunosuppressive effects include diminished bacterial defenses, G. W. Fischer et al., Lancet ii: 819-820, 1980, depressed reticuloendothelial system function, Z. Friedman et al, Pediatrics 61: 694-698, 1978, and inhibition of the lymphoproliferative responses which are a component of cell-mediated immune responses, S. Ladisch et al, Clin. Immunol. Immunopath. 25: 196-202, 1982. The cause of these immunosuppressive effects has not been identified.
These examples of detrimental clinically important side effects of lipid emulsions, and the recommendations of restrictions on the clinical use of the emulsions, clearly define a problem with the lipid emulsions and suggest that it is of great urgency that steps should be taken to avoid these adverse side effects. However, despite wide awareness of the serious side effects of the lipid emulsions as currently formulated, no solution to the problem has been found. It is this serious problem which is addressed by the present invention, which proposes that, by modification of the composition of the lipid emulsions, the immunosuppressive effects of these emulsions will be abrogated.
Specific background for understanding my invention claimed herein is derived from my studies on the mechanism by which lipid emulsions suppress one of the component steps of cell mediated immune responses, the proliferation of normal lymphocytes in response to stimulation by an antigen (such as a bacterial antigen). This proliferative phase of the cellular immune response is the normal division of lymphocytes (present in the blood), when they are exposed to such foreign antigens. Adequate division of the cells is necessary to ultimately result in an adequate immunologic response which will counteract an infectious process or eliminate foreign cells. Therefore, any agent which is immunosuppressive may interfere with the resolution of infections, the rejection of foreign cells, and possibly the rejection of tumor cells. My studies of the mechanism of the immunosuppressive effect of lipid emulsions have shown that the T lymphocyte is prevented from proliferating, and thereby is adversely affected, by the lipid emulsion. I have also found that such lipid emulsions cause inhibition of proliferation of essentially all other types of cells tested. In studying the mechanism of the inhibition of cell proliferation by lipid emulsions, using tumor cells for these studies, I found that this agent affects the post-DNA synthetic phase of cell division. This phase, known as the G.sub.2 phase, represents the period of time during which the cell increases its volume and total amount of membrane, to be able to divide into two daughter cells.
The present invention relates to the modification of the formulation of lipid emulsions such as Intralipid .RTM. and Liposyn .RTM. to abrogate the immunosuppressive effects which have been observed. As previously stated, the known lipid emulsions consist of three components: triglyceride, glycerol, and lecithin. In the absence of decomposition of the lipids contained in the lipid emulsions, I have found that the lecithin in the lipid emulsions accounts for the inhibitory effect, in the following fashion:
Lecithin and cholesterol exist in the cell membrane in equimolar amounts. Also, certain lipids, especially cholesterol, exchange freely between cells and their environment. I therefore hypothesized that because the commercially available, clinically used, lipid emulsions contain essentially no cholesterol, these lipid emulsions withdraw cholesterol from the membranes of cells exposed to the lipid emulsion by a process of passive exchange. Passive exchange acts to equalize the ratio of cholesterol to lecithin in the lipid emulsion (infinitely low) with that of the cell membranes (approximately 1:1 molar). Using radiolabelled lecithin and cholesterol, I have proved the correctness of this hypothesis by showing that cells in the presence of one of the lipid emulsions, Intralipid .RTM., lose cholesterol from their membranes. The significance of this finding is that adequate membrane cholesterol content is necessary for the cell to progress through the G.sub.2 phase of the cell cycle and to divide. My findings therefore provided an explanation for the inhibitory effects of the clinically used lipid emulsions. That is, these lecithin-containing, cholesterol-free emulsions of vegetable oils withdraw cholesterol from the cell membrane. By depleting the membrane cholesterol content the lipid emulsions thereby inhibit cell division, arresting the proliferative process in the G.sub.2 phase of the cell cycle.
The present invention is based on my ultimate finding that, to my surprise, by changing the composition of lipid emulsions only by adding a small amount of cholesterol to the triglyceride and lecithin comprising the known lipid emulsions, the immunosuppressive effects of the emulsions are abrogated, by preventing depletion of cell membrane cholesterol content by the lipid emulsion.